Changes in soil water content are known to affect soil reflectance. Even though it was suggested some time ago that the phenomenon of increased forward scattering due to the presence of water in the soil is related to water film thickness and matric potential, there has been no detailed investigation of this in any studies. The effects of moisture conditions on visible near-infrared (vis-NIR) spectra of four representative soils in Denmark have been assessed as a function of both water film thickness (expressed as the number of molecular layers) and matric potential. Complete water retention curves, from wet (pF 0.3, pF = log(|j|), where φ is the matric potential in cm) to hyper dry end (oven-dried and freeze-dried soil), were obtained by initial wetting followed by successive draining and drying of soil samples, performing NIR measurements at each step. Soil reflectance was found to decrease systematically, yet not proportionally, with decreasing matric potential and increasing molecular layers. The changes in molecular layers were best captured by the soil reflectance of clay-rich soils. Here the largest increase in reflectance occurred between pF 3 and 4, caused by the shift from capillary to adsorptive surface forces. In support of this, the smallest changes in reflectance were seen in the sandiest soil. Freeze drying the soil highest in organic C increased reflectance, possibly due to an alteration in organic matter during freezing. The different reflectance behavior of soil with a higher organic C content may be linked to differences in the amount, but also the quality (higher hydrophobicity) of the organic matter. However, this needs to be confirmed in further studies. Copyright © 2014. Copyright © by the Soil Science Society of America, Inc.